The overall goals of Project 2 are 1) to study the mechanisms responsible for the efficacy of the HSVtk/GCV system in eradicating tumors and 2) to optimize and further characterize the treatment of malignant mesothelioma using the Herpes Simplex thymidine kinase (HSVtk) gene carried in recombinant viral vectors. Preliminary data indicate that administration of a "first generation" adenovirus (Ad) vector containing the Herpes Simplex thymidine kinase (HSV) gene driven from a Rous Sarcoma Virus(RSV) promoter (Ad.RSVtk), in conjunction with the antiviral drug gancichovir (GCV), can eradicate established tumor in a model of human malignant mesothelioma growing within the peritoneal cavity of immunodeficient mice. This process appears to depend on a powerful "bystander effect" whereby only a relatively small percentage of cells need to be transduced with the Ad.RSVtk gene. These experiments suggest that this system may be of efficacy in treating certain human cancers, such as malignant mesothelioma and others that grow in defined anatomic spaces. Before such a goal is reached, however, a number of important questions about the mechanisms by which tumor eradication is effected , the optimal mode of delivery, and the role of the immune system must be answered. In addition, it will be critical to optimize the system by evaluating the ability of improved viral vectors containing HSVtk\generated in Project 1 to treat tumor. A major focus of this proposal will be to study possible mechanisms by which the HSVtk/GCV system eradicates tumor. Experiments examining the role of the role of the immune system, vascular system, and gap junctional transfer of toxic metabolites will be conducted. The treatment of malignant mesothelioma using the first generation Ad.RSVtk vector will be further characterized and optimized by developing and evaluating models of pleural mesothelioma in immunodeficient (nude) rats and immunocompetent animals (Fischer rat). Finally, the efficacy of second generation adenoviruses containing HSVtk and newly developed viral vectors containing hSVtk generated in Project 1 in treating models of mesothelioma in immunodeficient and immunocompetent animals will be studied. The information gained about he mechanisms and optimization of the HSVtk/GCV system and the testing of new vectors designed in Project 1 (i.e. adeno- associated virus) will be critical in designing and implementing new clinical trials in Project 3.